Project Summary Regulatory small RNAs (sRNAs) play important roles in stress responses in nearly all bacterial organisms that have been investigated, including pathogenic bacteria. The functions of sRNAs are often supported by proteins such as the paradigmatic bacterial RNA chaperone protein, Hfq. While much is known about the mechanism of Hfq-RNA interactions, significant gaps remain in our understanding of how other bacterial RNA chaperones ? e.g the proteobacterial RNA-binding protein ProQ ? interact with RNA to regulate gene expression. Further, traditional approaches to discover RNA-binding proteins have left many bacterial species without an identified RNA chaperone protein: almost half of bacterial genomes contain neither an hfq nor proQ ortholog despite many of these organisms, including important human pathogens, being known to utilize sRNAs. Given the instability of unbound, untranslated RNA within a bacterial cell, this suggests that there are one or more as-of-yet undiscovered bacterial RNA chaperone proteins. This proposal builds on a recently-developed genetic approach to probe RNA-protein interactions inside of E. coli cells with a transcription-based bacterial three-hybrid (B3H) assay. This assay can detect interactions in the native context of a bacterial cytoplasm and offers a genetic approach to identify novel RNA-binding proteins, as well as to identify and interrogate mutations in these proteins with molecular phenotypes of interest. The long-term goal of the PI?s laboratory is to understand the molecular mechanisms by which RNA chaperones interact with sRNAs to drive bacterial gene regulation. This proposal aims to define the mechanism of ProQ-RNA interactions as well as to identify novel RNA chaperones. Aim 1 will develop a second-generation B3H reporter with improved sensitivity that will be generalizable to many RNA-protein interactions. Aim 2 will utilize forward and reverse molecular genetics to probe the specificity-determinants of ProQ-RNA interactions, identifying critical contributions of the structure of both the protein and its target RNAs. Aim 3 will screen libraries of genomic fragments from bacterial pathogens such as Mycobacterium tuberculosis to identify novel RNA-binding proteins that interact with sRNAs expressed by these organisms. The proposed research is innovative because it approaches the discovery and analysis of bacterial RNA-binding proteins through development and application of a novel genetic methodology with the potential to become a useful tool for many in the field. In addition, the knowledge gained in the identification of new candidate RNA-chaperone proteins as well as the mechanism of ProQ-RNA interactions will increase the potential of bacterial RNA chaperones to serve as therapeutic targets for bacterial infections.